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Human knee-joint cartilage cell in-vitro amplification method for clinic treatment
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A chondrocyte, in vitro expansion technology, applied in the medical field, can solve the problems of slow expansion of in vitro chondrocytes, lack of safety, in vitro chondrocytes not suitable for clinical use, etc.
Inactive Publication Date: 2014-06-25
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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[0004] The purpose of the present invention is to provide a clinically applicable human knee jointchondrocytein vitro expansion method to solve the problem that in vitro chondrocytes in the prior art cannot be applied clinically, and the expansion speed of in vitro chondrocytes is relatively slow and lacks safety. technical issues
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Embodiment 1
[0026] 1. Chondrocyte extraction, isolation, culture and identification
[0027] 1. Chondrocyte extraction, isolation and culture
[0028] (1) Separation of serum from patients
[0029] Collect 40-50ml of venous blood from the patient, put it in 40-50ml of normal saline containing 10u / ml heparin, centrifuge at 3000-3500 rpm for 10-20 minutes, extract the upper serum, filter it with a 0.1 micron filter, and store it at 4°C.
[0030] (2) Collection of articular cartilage in the non-weight-bearing area of the patient
[0031] Arthroscopy was used to extract 100-200 mg of cartilage from the non-weight-bearing area of the patient's joint. After routine disinfection, draping, and local anesthesia, the cartilage was removed under arthroscopy and placed in 9 g / L cold sterile saline.
[0032] (3) Extraction and separation of chondrocytes
[0033] Chopped cartilage into 0.8~1.2mm 3 size, washed 3 times with culture medium (Ham's F12 medium, containing 10mmol / L HEPES buffer, 70μmo...
Embodiment 2
[0043] 1. Chondrocyte extraction, isolation, culture and identification
[0044] 1. Chondrocyte extraction, isolation and culture
[0045] (1) Separation of serum from patients
[0046] Collect 40-50ml of venous blood from the patient, place it in 40-50ml of normal saline containing 10u / ml heparin, centrifuge at 3000-3500 rpm for 10-20 minutes, extract the upper serum, filter it with a 0.1 micron filter, and store it at 4°C.
[0047] (2) Collection of articular cartilage in the non-weight-bearing area of the patient
[0048] 150 mg of cartilage in the non-weight-bearing area of the patient's joint was collected by arthroscopy. After routine disinfection, draping, and local anesthesia, the cartilage was removed under arthroscopy and placed in 9 g / L cold sterile saline.
[0049] (3) Extraction and separation of chondrocytes
[0050] Chopped cartilage into 0.8~1.2mm 3 size, washed 3 times with culture medium (Ham's F12 medium, containing 10mmol / L HEPES buffer, 70μmol / L g...
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Abstract
The invention discloses a human knee-joint cartilagecell in-vitro amplification method for clinic treatment. The method comprises the following steps: separating and extracting in-vitro cartilage of a patient, and obtaining cartilage cells; inoculating the cartilage cells to a culture medium for performing primary cell culture; performing cell subculture when the confluence degree of the cartilage cells subjected to primary cell culture is 70-80 percent, wherein the culture medium for performing primary cell culture and cell subculture comprises patient in-vitro serum with the volume fraction of 5-20 percent, 20-40ng / ml of FGF and 20-40ng / ml of PDGF. The method is safe and reliable, and the cartilage cells obtained through culture are suitable for clinical use.
Description
technical field [0001] The invention relates to a cell in vitro culture technology in the medical field, which is suitable for the treatment of articular cartilage defects in orthopedics, and particularly relates to an in vitro expansion method for human knee articular chondrocytes that can be used clinically. Background technique [0002] Chondrocytes are the only cell type in articular cartilage, and they are terminal cells that lack the ability to proliferate in the internal environment of synovial fluid. In addition, articular cartilage has no blood supply, and once damaged, it lacks the ability to regenerate and repair. At present, the methods for treating human cartilage diseases include the method of transplanting cartilage tissue collected from other parts of the autologous body to the defect site for treatment. This method has the problems of limited selection of collection sites and collection volume, and cannot be effectively applied. For this reason, those skille...
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